The formation of heterochromatin is important not only for chromosome structure and segregation but is involved in biological processes that can lead to cancer and developmental diseases. Heterochromatin is often initiated at telomeres and centromeres and can spread into adjacent euchromatin. Understanding the molecular mechanisms by which yeast heterochromatin initiates, spreads and meets boundaries to spreading are the goals of this proposal. These mechanisms involve the post-translational modifications of histones both inside and outside of heterochromatin. The role of histone H3 K36 methylation in heterochromatin will be studied in its ability to initiate and propagate heterochromatin. Conversely, the acetylation of histone H4 lysine 16 and methylation of histone H3 lysine 79 in euchromatin will be investigated in preventing the spread of heterochromatin. Also, new heterochromatin-like HAST domains will be investigated to determine how they form, spread, repress gene activity and retard the firing of replication origins. This work will involve the use of genetics, chromatin immunoprecipitation and biochemical analysis to identify factors regulating the initiation and spreading of heterochromatin. The work will also involve the use of genome wide histone acetylation, methylation and protein binding studies to determine genome wide mechanisms of heterochromatin formation and to search for novel forms of heterochromatin. Given that a number of proteins affecting heterochromatin are conserved from yeast to humans, these studies are expected to identify new strategies for treating human disease states.